 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jun 27, 2021 |
| Title |
BAP1-KO-clone2-rep2 |
| Sample type |
SRA |
| |
|
| Source name |
mESC
|
| Organism |
Mus musculus |
| Characteristics |
sample type: BAP1-KO-clone2-rep2
|
| Treatment protocol |
Indicated cells were treated for 48 hours with 500nM dTAG-13 (TOCRIS), or DMSO as vehicle, in order to degrade dTAG-BAP1. To generate stable KO cell lines, 10ug pX458 2.0 plasmid pairs (Addgene) encoding Cas9 and sgRNAs were transfected using Lipofectamine 2000 (Invitrogen), according to manufacturer’s instruction. Sorting of GFP positive cells was carried out 48 hours after transfection and 1000 cells were seeded onto a 15-cm dish. Clones were isolated 10-14 days later and grown further before screening for knockout by Western blot. For rescue clone generation, mESCs were transfected with 10ug pCAG vectors encoding 2xFlag-HA-tagged BAP1 wild-type or BAP1 C91S using Lipofectamine 2000 (ThermoFisher Scientific), according to manufacturer’s instructions. 24 hours post-transfection puromycin selection (1µg/ml) was added for a further 24 hours. Cells were then split to clonal density (~1:40) onto a 15cm plate. Clones were isolated 10-14 days later and grown further before screening for rescue allele expression by Western blot.
|
| Growth protocol |
mESCs were grown on 0.1% gelatin-coated dishes in 2i/LIF-containing GMEM medium (Euroclone) supplemented with 20% fetal calf serum (Euroclone), 2 mM glutamine (Gibco), 100 U/ml penicillin, 0.1 mg/ml streptomycin (Gibco), 0.1 mM non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 50 µM ß-mercaptoethanol phosphate buffered saline (PBS; Gibco), 1000 U/ml leukemia inhibitory factor (LIF; produced in-house), and GSK3β and MEK 1/2 inhibitors (ABCR GmbH) to a final concentration of 3 mM and 1 mM, respectively.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted with the Quick-RNA™ MiniPrep extraction kit (Zymo Research) and retro-transcribed with ImProm-II™ Reverse Transcription System (Promega) according to the manufacturer’s instructions
RNA-seq was performed following SMART-seq2 protocol (Picelli et al., 2014)
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Alignment; STAR v2.7; --outSAMattributes Standard --outFilterMultimapNmax 1
Feature-counting; featureCounts; -s 0 -t exon -g gene_name
Differential Gene Expression Analysis; DESeq2 v1.24; default parameters
mm10
WT-rep1.rpkm: Gene Expression Analysis (RPKM); WT-rep2.rpkm: Gene Expression Analysis (RPKM); BAP1-KO-clone1-rep1.rpkm: Gene Expression Analysis (RPKM); BAP1-KO-clone2-rep1.rpkm: Gene Expession Analysis (RPKM); BAP1-KO-clone2-rep2.rpkm: Gene Expression Analysis (RPKM); BAP1-KO-clone1-vs-WT_diffexp_log2fc1.5_pval0.05.tsv: Differential Expression Analysis; BAP1-KO-clone1-vs-WT_diffexp_log2fc1.5_pval0.05.tsv: Differential Expression Analysis
|
| |
|
| Submission date |
Dec 06, 2020 |
| Last update date |
Jun 27, 2021 |
| Contact name |
Federico Rossi |
| E-mail(s) |
federico.rossi@ieo.it
|
| Organization name |
Istituto Europeo Oncologia
|
| Street address |
Via Adamello 16
|
| City |
Milan |
| ZIP/Postal code |
10142 |
| Country |
Italy |
| |
|
| Platform ID |
GPL24247 |
| Series (1) |
| GSE162739 |
BAP1 activity regulates Polycomb occupancy and global chromatin condensation counteracting diffuse PCGF3/5-dependent H2AK119ub1 deposition |
|
| Relations |
| BioSample |
SAMN17011930 |
| SRA |
SRX9637880 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4958886_BAP1-KO-clone2-rep2.rpkm.gz |
345.8 Kb |
(ftp)(http) |
RPKM |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
